This invention relates to processes for producing acid-soluble short-chain peptides from a protein material, to the short-chain peptides produced thereby, and to edible compositions containing these short-chain peptides.
Many foods and beverages are fortified with supplemental protein derived from various plant and animal sources to increase the nutritional value of the food or beverage. Commonly used inexpensive supplemental protein sources include vegetable protein materials such as soybeans, legumes, oilseeds, and rapeseed, and animal protein materials such as casein.
Unmodified protein materials derived from such protein sources cannot be used effectively as a supplement in certain beverages and food compositions in which nutritional fortification is desirable. One such group of beverages in which protein fortification is desirable are acidic beverages such as soft drinks, carbonated soft drinks, juices, and nutritional sports beverages. Acidic beverages, however, have pH levels at which unmodified protein materials are generally insoluble. Unmodified protein materials added to such acidic beverages cause undesirable cloudiness and sedimentation.
Another group of compositions in which protein fortification is desirable but not practicible with unmodified protein materials are enteral compositions for providing nutrition to persons having impaired digestive function. Persons having impaired digestive function are often incapable of fully absorbing and digesting unmodified protein, therefore effective enteral compositions cannot be formed using such protein materials.
It is known in the art to modify supplemental protein materials by hydrolyzing the protein to form peptides which are useful as nutritive components of foods and beverages in which unmodified proteins cannot be utilized. Protein hydrolyzates are commonly used to form nutritional acidic beverages since protein hydrolyzates are more soluble in acidic aqueous solutions than unmodified proteins, and are used to form enteral compositions since the protein hydrolyzate peptides are more easily absorbed and digested than unmodified proteins.
Protein materials are often hydrolyzed by treatment with a proteolytic enzyme under conditions at which the enzyme hydrolyzes the protein into peptides of intermediate length. Intermediate length peptides are generally more soluble in acidic solutions than unmodified proteins and have been used to form nutritionally enhanced acidic beverages.
Hydrolysis of the protein material into short-chain peptides is conventionally avoided to prevent the formation of "off-flavor" or "bitter" materials. For example, U.S. Pat. No. 3,897,570 to Yokotsuka et al. provides a process for producing an acid soluble protein beverage in which a slurry is formed of a protein material, the slurry is heated by steam under pressure to denature the protein in the slurry, the protein is hydrolyzed with an acid protease at a pH of 2.5-6.0 so the ratio of formol state nitrogen to total nitrogen in the filtrate of the slurry is less than 20% to prevent over-decomposition of the protein, and the slurry is filtered to separate a solution containing acid soluble peptides from insoluble residual materials. Other such processes for producing an acidic protein beverage are disclosed in U.S. Pat. Nos. 3,846,560 and 3,876,806 to Hempenius et al. in which a slurry of protein material is hydrolyzed with a proteolytic enzyme and the hydrolysis is terminated before it proceeds to the point of producing a significant amount of products which cause a bitter taste.
Enzymatic hydrolysis of protein materials is often conducted under conditions which facilitate the formation of intermediate length peptides. Frequently, enzymatic hydrolysis is conducted on protein materials which are partially to substantially solubilized in a solution containing the enzymes. Solubilized protein material is more easily accessible to the proteolytic enzymes than insoluble protein material since a substantial portion of the solubilized protein is exposed to enzymic activity. Intermediate length peptides are easily formed from a solubilized protein material since the enzymes can cleave centrally located peptide bonds in the protein which would not be exposed to enzymic activity if the protein material were insoluble in the solution.
The solubility of a protein material in a solution is determined by the pH of the solution relative to the isoelectric point of the protein material. Protein materials are least soluble at or near the isoelectric point of the protein, which typically is a pH which is moderately acidic (e.g. soy protein has an isoelectric point at about pH 4.5, and casein has an isoelectric point at about pH 4.7). The solubility of a protein material in a solution increases as the pH of the solution moves away from the isoelectric point of the protein, therefore, protein materials tend to be more soluble in basic, neutral, and strongly acidic mediums.
Several processes are known for enzymatically hydrolyzing protein materials under neutral, basic, or strongly acidic pH conditions. Often the pH conditions of the proteolysis are selected to optimize the activity of the enzyme rather than to determine length of the resulting peptides. For example, U.S. Pat. No. 4,107,334 to Jolly provides that the pH of the enzymatic hydrolysis depends on the nature of the protein to be hydrolyzed and the proteolytic enzyme employed, and is selected to optimize the conversion of the protein to functional protein, and then provides several examples in which enzymatic protein hydrolysis is effected at pHs of 2.5 and 7.0-9.5. U.S. Pat. No. 4,687,739 to Sugisawa et al., and U.S. Pat. Nos. 3,876,806 and 3,846,560 to Hempenius et al. disclose processes for enzymatically hydrolyzing a protein material at a neutral pH. U.S. Pat. Nos. 3,741,771 and 3,713,843 to Pour-El provide processes for enzymatically hydrolyzing a protein material under strongly acidic pH conditions.
Another factor which facilitates the formation of intermediate length peptides from a protein material, as opposed to short-chain peptides, is the type of enzyme commonly selected for hydrolyzing the protein material. Enzymes commonly employed to hydrolyze a protein material have primarily endopeptidase (or "protease") activity. Endopeptidases cleave a protein material between amino acids located in the interior of the protein, and produce two peptides of relatively substantial molecular weight each time the endopeptidase cleaves the protein material (as compared to exopeptidases which cleave amino acids at or near the end of the protein and produce a large molecular weight peptide and a small molecular weight peptide or an amino acid). Endopeptidases are commonly employed in protein hydrolysis since the endopeptidases rapidly cleave the protein material into smaller, but still substantial, peptides which are more acid soluble than intact protein, and may be more easily absorbed and digested than intact protein. For example, U.S. Pat. No. 4,687,739 teaches that soybean protein is enzymatically hydrolyzed in the process with an endopeptidase, U.S. Pat. No. 3,897,570 provides that any acid protease may be used in the process to hydrolyze soybean protein, U.S Pat. No. 3,713,843 discloses that the enzymes useful to treat proteins in the process are enzymes which are active in breaking the peptide bond, and U.S. Pat. No. 4,107,334 provides that any acid, neutral, or alkaline protease is suitable for use in the process.
Non-bitter short-chain peptides, however, are more desirable than intermediate length peptides in numerous applications, including acidic beverages and enteral compositions. Short-chain peptides are more soluble in an acidic medium, and do not tend to settle out of solution as some intermediate length peptides do. Therefore, short-chain peptides are very useful for forming acidic nutritional beverages which are not subject to cloudiness or precipitation. Short-chain peptides are more desirable in enteral compositions than intermediate length peptides because short-chain peptides are more easily absorbed and digested than intermediate length peptides.
European Patent Application No. 0 461 261 A1 provides a short-chain peptide composition which is rich in branched chain amino acids and low in aromatic amino acid content, and a method of making the composition. The application discloses that the composition is formed by hydrolyzing an animal or a plant protein with an enzyme composition having exopeptidase activity and endopeptidase activity, where the exopeptidase activity is used to lower the aromatic amino acid content.
In the process of EP 0 461 261 A1, the enzymolysis of the protein is conducted only at neutral pH--which results in a peptide composition having a varied peptide chain-length profile, since the protein is at least partially soluble at neutral pH, and is likely to be cleaved into intermediate length peptides by the endopeptidase enzymes. These intermediate length peptides are subject to reaction with the exopeptidases (as well as further cleavage by endopeptidases) and are likely shorter in peptide chain length than the intermediate length peptides formed according to the processes discussed above. However, due to the enzymolysis at neutral pH, intermediate chain length peptides are still likely to be present in the resulting composition to an appreciable extent.
U.S. Pat. No. 3,830,942 to Hawley provides a soluble protein product for use in highly acid foods, and a process for making the same. An aqueous slurry is formed of a vegetable protein or soy protein isolate having a pH at approximately the isoelectric point of the protein. The slurry may be heated to make the protein more susceptible to the action of the enzymes. The protein is then hydrolyzed with any enzyme effective in the pH range of the isoelectric point of the protein. The resulting peptides are shorter in length than intermediate length peptides formed by enzymatic hydrolysis at pH levels in which the protein material is soluble since enzymatic hydrolysis is confined only to insoluble protein material exposed to the enzymes. However, due to the use of enzymes which have predominately or exclusively endopeptidase activity, the resulting composition contains an appreciable amount of intermediate length peptides.
It is desirable to produce an enzymatically hydrolyzed protein material having a narrow range of peptide chain lengths where a significant quantity of the peptides have a chain length of 7 peptides or less. Such an peptide material is of particular use in acidic beverages and enteral compositions.